Ultra-high-sensitive assay of protein and nucleic acid and kit, and novel enzyme substrate

ABSTRACT

Provided is a ultra-high-sensitivity assay in which the assay can be made on a commonly used assay apparatus such as an absorptiometer and a plate reader or with naked eyes. The high-sensitivity assay in which the assay can be made on a commonly used assay apparatus or with naked eyes can be provided by combining an enzyme cycling method using thio-NAD(P) as a coenzyme, a labeling enzyme and a substrate for the labeling enzyme optimally, and by amplifying thio-NAD(P)H, which is a signaling substance, exponentially and then quantifying the thio-NAD(P)H colorimetrically.

CROSS-REFERENCE TO RELATED PATENT APPLICATION

The present application claims priority under Japanese PatentApplication 2011-63559, filed on Mar. 23, 2011, the entire contents ofwhich are hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates to a high-sensitive assay of protein andnucleic acid and a kit using an enzyme immunoassay and a nucleic acidprobe assay. More particularly, the present invention relates to amethod enabling ultra-high-sensitive assay of protein or nucleic acidwhile avoiding interference caused in thio-NAD cycling method used inthese enzyme activity assay by employing an enzyme substrate specific toan enzyme labeled to an antibody or nucleic acid probe. The presentinvention further relates to novel enzyme substrates which can be usedin the above high-sensitive assay of protein and nucleic acid and a kit.

BACKGROUND TECHNOLOGY

Although the radioimmunoassay (RIA) method is technically established asa high sensitivity measurement of protein or nucleic acid, under thepresent circumstances, it is impossible to change the aforementionedmethod to one having high sensitivity beyond a 10⁻¹⁶ moles degreebesides improvement of the sensitivity of a detection equipment. And bythe RIA method, the place of measurement is not only limited to anisotope experimental facility, but the expiration date of reagents willbecome extremely short and the sensitivity of reagents will be decreaserapidly, because of using the nuclide of a short time. It also has theproblem of radioactive waste problem to the method of radiationmeasurement. Especially the problem of abandonment in the case of usinga long lasting nuclide is serious. Therefore, the research anddevelopment of the high sensitivity measurement of protein or nucleicacid have been developed considering “non-radioactive” as a keywordrecently. Thus most of the research and development and the technicalimprovement about the RIA method is not performed in the actualconditions.

A high sensitivity measurement replaced with the RIA method, which isenzyme immunoassay (ELISA method) in the measurement of protein (FIG.1), and PCR method in the measurement of nucleic acid. The enzymeimmunoassay can be proceed to high sensitivity (10⁻¹⁵ moles) by thefluorescence method and the emitting light method from sensitivity of10⁻¹³ mole by the colorimetric assay in the initial development, and thedevelopment and improvement of the exclusive measurement device are alsoproceeding. However, the sensitivity has been arrived to limit, just theoperation of measurement is simple.

Moreover, in the PCR method of the high sensitivity measurement ofnucleic acid, it is considered the problem of the detection of specificsignal for target molecule, the amplification efficiency and thecondition of PCR product arriving at a plateau, the quantification ofnucleic acid is difficult strictly.

The enzyme immunoassay using antibody-enzyme complex and the nucleicacid probe assay using a nucleic acid-enzyme complex using thio-NADcycling assay is already known. (Refer to WO2008/117816 (Patent document1))

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

According to method of Patent document 1, the enzyme immunoassay wascombined with the enzymatic cycling method which uses as a substrateproducing by the labeling enzyme using the enzyme immunoassay, andthio-NAD(P)H as a signal substance is amplified geometric-progressive,and by a colorimetric assay method, it results in the quantification ofprotein or nucleic acid and the detection by nucleic acid. However itrevealed that not necessarily high reactivity of the labeled enzyme andsubstrate, and the substrate of labeled cycling enzyme partiallyinhibits the enzyme reaction in the enzymatic cycling reaction.

The object of the present invention is in the enzyme immunoassay, themethod of being combined with the cycling method which use as asubstrate producing by the labeled enzyme used in enzyme immunoassay,solving the aforementioned problem of the substrate to labeled enzyme,and using a colorimetric assay method, high sensitivity detection methodof protein or nucleic acid by colorimetric assay of the easiest method,and providing the assay of increasing the sensitivity togeometric-progressive degree. In particular, it is provided the assaymethod which raised sensitivity more than 10⁻¹⁸ moles.

The present inventor has succeeded in obtaining a compound as asubstrate to labeled enzyme solving the aforementioned problem orproviding the new compound, and therefore using these substrates, he hassucceeded in solving the aforementioned problem, thus the presentinvention has been completed.

Means for Solving the Problems

The present invention is as follows:

-   [1] A method of assaying enzyme activity using an antibody-enzyme    complex, wherein

alkaline phosphatase, glucosidase, galactosidase, fructosidase,mannosidase or peroxidase is used as an enzyme of the antibody-enzymecomplex, and

an androsterone derivative represented by the following formula (1) isused as a substrate of the enzyme,

wherein

(i) X represents a phosphate group, and Y¹ and Y² represent together amethylene group, or Y¹ represents hydrogen, and Y² represents hydrogen,a hydroxyl group, a C₁₋₆ alkoxy group, or a C₁₋₆ alkyl group when theandrosterone derivative represented by the formula (1) is used as asubstrate of alkaline phosphatase,

(ii) X represents a sugar moiety, the sugar moiety represents oneselected from the group consisting of glucose, galactose, fructose andmannose, and Y¹ and Y² represent together a methylene group or an oxygenatom, or Y¹ represents hydrogen, and Y² represents hydrogen, a hydroxylgroup, a C₁₋₆ alkoxy group, or a C₁₋₆ alkyl group when the androsteronederivative represented by the formula (1) is used as a substrate ofglucosidase, galactosidase, fructosidase or mannosidase, or

(iii) X represents —O—CO—R (provided that R represents a C₁₋₆ alkylgroup or a phenyl group), Y¹ and Y² represent together a methylene groupor an oxygen atom, or Y¹ represents hydrogen, and Y² representshydrogen, a hydroxyl group, a C₁₋₆ alkoxy group, or a C₁₋₆ alkyl groupwhen the androsterone derivative represented by the formula (1) is usedas a substrate of peroxidase, and

a quantification of a product of the enzyme reaction is performed byproducing thio-NADH and/or thio-NADPH by enzyme cycling reaction usingNADH and/or NADPH, thio-NAD and/or thio-NADP, and hydroxysteroiddehydrogenase (HSD), and assaying the amount of the produced thio-NADHand/or thio-NADPH, or measuring the change of the color by the producedthio-NADH and/or thio-NADPH.

-   [2] A method of assaying a nucleic acid probe using an    enzyme-labeled nucleic acid probe, wherein

alkaline phosphatase, glucosidase, galactosidase, fructosidase,mannosidase or peroxidase is used as an enzyme of the enzyme-labelednucleic acid probe, and

an androsterone derivative represented by the following formula (1) isused as a substrate of the enzyme,

wherein

(i) X represents a phosphate group, and Y¹ and Y² represent together amethylene group, or Y¹ represents hydrogen, and Y² represents hydrogen,a hydroxyl group, a C₁₋₆ alkoxy group, or a C₁₋₆ alkyl group when theandrosterone derivative represented by the formula (1) is used as asubstrate of alkaline phosphatase,

(ii) X represents a sugar moiety, the sugar moiety represents oneselected from the group consisting of glucose, galactose, fructose andmannose, and Y¹ and Y² represent together a methylene group or an oxygenatom, or Y¹ represents hydrogen, and Y² represents hydrogen, a hydroxylgroup, a C₁₋₆ alkoxy group, or a C₁₋₆ alkyl group when the androsteronederivative represented by the formula (1) is used as a substrate ofglucosidase, galactosidase, fructosidase or mannosidase, or

(iii) X represents —O—CO—R (provided that R represents a C₁₋₆ alkylgroup or a phenyl group), Y¹ and Y² represent together a methylene groupor an oxygen atom, or Y¹ represents hydrogen, and Y² representshydrogen, a hydroxyl group, a C₁₋₆ alkoxy group, or a C₁₋₆ alkyl groupwhen the androsterone derivative represented by the formula (1) is usedas a substrate of peroxidase, and

a quantification of a reaction product of the enzyme reaction isperformed by producing thio-NADH and/or thio-NADPH by enzyme cyclingreaction using NADH and/or NADPH, thio-NAD and/or thio-NADP, andhydroxysteroid dehydrogenase (HSD), and assaying the amount of theproduced thio-NADH and/or thio-NADPH, or measuring the change of thecolor by the produced thio-NADH and/or thio-NADPH.

-   [3] The method according to [1] or [2], wherein

the enzyme is alkaline phosphatase, and

X represents a phosphate group, Y¹ and Y² represent together a methylenegroup, or Y¹ represents hydrogen, and Y² represents hydrogen, a hydroxylgroup, a C₁₋₆ alkoxy group, or a C₁₋₆ alkyl group in the androsteronederivative represented by the formula (1).

-   [4] The method according to [1] or [2], wherein

the enzyme is glucosidase, galactosidase, fructosidase or mannosidase,and

X represents a sugar moiety, the sugar moiety represents one selectedfrom the group consisting of glucose, galactose, fructose and mannose,and Y¹ and Y² represent together a methylene group or an oxygen atom, orY¹ represents hydrogen, and Y² represents hydrogen, a hydroxyl group, aC₁₋₆ alkoxy group, or a C₁₋₆ alkyl group in the androsterone derivativerepresented by the formula (1).

-   [5] The method according to [1] or [2], wherein

the enzyme is peroxidase, and

X represents —O—CO—R (provided that R represents a C₁₋₆ alkyl group or aphenyl group), and Y¹ and Y² represent together a methylene group or anoxygen atom, or Y¹ represents hydrogen, and Y² represents hydrogen, ahydroxyl group, a C₁₋₆ alkoxy group, or a C₁₋₆ alkyl group in theandrosterone derivative represented by the formula (1).

-   [6] A kit for enzyme immunoassay comprising reagents (1) to (5)    described below:

(1) alkaline phosphatase labeled with an antibody specific to a targetprotein antigen,

(2) an androsterone derivative represented by the formula (1), which isa substrate of the enzyme described above

(wherein, X represents a phosphate group, Y¹ and Y² represent together amethylene group, or Y¹ represents hydrogen, and Y² represents hydrogen,a hydroxyl group, a C₁₋₆ alkoxy group, or a C₁₋₆ alkyl group),

(3) hydroxysteroid dehydrogenase (HSD),

(4) NADH and/or NADPH, and

(5) thio-NAD and/or thio-NADP.

-   [7] A kit for enzyme immunoassay comprising reagents (1) to (5)    below:

(1) glucosidase, galactosidase, fructosidase or mannosidase which islabeled with an antibody specific to a target protein antigen,

(2) an androsterone derivative represented by the formula (1), which isa substrate of the enzyme described above

(wherein X represents a sugar moiety, the sugar moiety represents oneselected from the group consisting of glucose, galactose, fructose andmannose, and Y¹ and Y² represent together a methylene group or an oxygenatom, or Y¹ represents hydrogen, and Y² represents hydrogen, a hydroxylgroup, a C₁₋₆ alkoxy group, or a C₁₋₆ alkyl group),

(3) hydroxysteroid dehydrogenase (HSD)

(4) NADH and/or NADPH, and

(5) thio-NAD and/or thio-NADP.

-   [8] A kit for enzyme immunoassay comprising reagents (1) to (5)    below:

(1) peroxidase labeled with an antibody specific to a target proteinantigen,

(2) an androsterone derivative represented by the formula (1), which isa substrate of the enzyme described above

(wherein X represents —O—CO—R (provided that R represents a C₁₋₆ alkylgroup or a phenyl group), Y¹ and Y² represent together a methylene groupor an oxygen atom, or Y¹ represents hydrogen, and Y² representshydrogen, a hydroxyl group, a C₁₋₆ alkoxy group, or a C₁₋₆ alkyl group),

(3) hydroxysteroid dehydrogenase (HSD),

(4) NADH and/or NADPH, and

(5) thio-NAD and/or thio-NADP.

-   [9] A kit for assaying a nucleic acid probe comprising reagents (1)    to (5) below:

(1) alkaline phosphatase labeled with a nucleic acid probe specificallybinding to a target nucleic acid,

(2) an androsterone derivative represented by the formula (1), which isa substrate of the enzyme described above

(wherein X represents a phosphate group, Y¹ and Y² represent together amethylene group, or Y¹ represents hydrogen, and Y² represents hydrogen,a hydroxyl group, a C₁₋₆ alkoxy group, or a C₁₋₆ alkyl group),

(3) hydroxysteroid dehydrogenase (HSD),

(4) NADH and/or NADPH, and

(5) thio-NAD and/or thio-NADP.

-   [10] A kit for assaying a nucleic acid probe comprising reagents (1)    to (5) below:

(1) glucosidase, galactosidase, fructosidase or mannosidase labeled witha nucleic acid probe specifically binding to a target nucleic acid,

(2) an androsterone derivative represented by the formula (1), which isa substrate of the enzyme described above

(wherein X represents a sugar moiety, the sugar moiety represents oneselected from the group consisting of glucose, galactose, fructose andmannose, and Y¹ and Y² represent together a methylene group or an oxygenatom, or Y¹ represents hydrogen, and Y² represents hydrogen, a hydroxylgroup, a C₁₋₆ alkoxy group, or a C₁₋₆ alkyl group),

(3) hydroxysteroid dehydrogenase (HSD),

(4) NADH and/or NADPH, and

(5) thio-NAD and/or thio-NADP.

-   [11] A kit for assaying a nucleic acid probe comprising reagents (1)    to (5) below:

(1) peroxidase labeled with a nucleic acid probe specifically binding toa target nucleic acid,

(2) an androsterone derivative represented by the formula (1), which isa substrate of the enzyme described above

(wherein X represents —O—CO—R (provided that R represents a C₁₋₆ alkylgroup or a phenyl group), Y¹ and Y² represent together a methylene groupor an oxygen atom, or Y¹ represents hydrogen, and Y² representshydrogen, a hydroxyl group, a C₁₋₆ alkoxy group, or a C₁₋₆ alkyl group),

(3) hydroxysteroid dehydrogenase (HSD),

(4) NADH and/or NADPH, and

(5) thio-NAD and/or thio-NADP.

-   [12] An androsterone derivative represented by the formula (1)    below:

(wherein, definitions of X, Y¹ and Y² are those of any one of (A), (B)and (C) below:

(A) X represents a phosphate group, Y¹ and Y² represent together amethylene group, or Y¹ represents hydrogen, and Y² represents a hydroxylgroup, a C₁₋₆ alkoxy group or a C₁₋₆ alkyl group,

(B) X represents one of a sugar moiety selected from the groupconsisting of glucose, galactose, fructose and mannose, Y¹ and Y²represent together a methylene group, or Y¹ represents hydrogen, and Y²represents hydrogen, a C₁₋₆ alkoxy group, or a C₁₋₆ alkyl group,

(C) X represents —O—CO—R, R represents a C₁₋₆ alkyl group or a phenylgroup, and Y¹ and Y² represent together a methylene group or an oxygenatom, or Y¹ represents hydrogen, and Y² represents hydrogen, a hydroxylgroup, a C₁₋₆ alkoxy group, or a C₁₋₆ alkyl group.).

-   [13] The androsterone derivative according to [12] wherein

the definitions of X, Y¹ and Y² are those of (A) X represents aphosphate group, and Y¹ and Y² represent together a methylene group, orY¹ represents hydrogen, and Y² represents a hydroxyl group, a C₁₋₆alkoxy group or a C₁₋₆ alkyl group.

-   [14] The androsterone derivative according to [12] wherein

the definitions of X, Y¹ and Y² are those of (B) X represents one of asugar moiety selected from the group consisting of glucose, galactose,fructose and mannose, and Y¹ and Y² represent together a methylenegroup, or Y¹ represents hydrogen, and Y² represents hydrogen, a C₁₋₆alkoxy group, or a C₁₋₆ alkyl group.

-   [15] The androsterone derivative according to [12] wherein

the definitions of X, Y¹ and Y² are those of (C) X represents —O—CO—R, Rrepresents a C₁₋₆ alkyl group or a phenyl group, and Y¹ and Y² representtogether a methylene group or an oxygen atom, or Y¹ represents hydrogen,and Y² represents hydrogen, a hydroxyl group, a C₁ alkoxy group, or aC₁₋₆ alkyl group.

Effects of the Invention

According to the present invention, it is possible to provide an enzymeimmunoassay enhancing the assay sensitivity to 10⁻¹⁸ moles or more, andto measure a target protein or target nucleic acid in high sensitivity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the assay principle of the enzyme immunoassay (ELISA method);

FIG. 2 is the calibration curve of Pumilio obtained in ComparativeExample 1;

FIG. 3 is the calibration curve of Pumilio obtained in ComparativeExample 2;

FIG. 4 is the calibration curve of Pumilio obtained in ComparativeExample 3;

FIG. 5 is the calibration curve of Pumilio obtained in ComparativeExample 4;

FIG. 6 is example compounds of some 3α-hydroxyandrostane.

FIG. 7 is the calibration curve of alkaline phosphatase obtained inExample 1;

FIG. 8 is the calibration curve of alkaline phosphatase obtained inExample 2;

FIG. 9 is the calibration curve of Pumilio obtained in Example 3;

FIG. 10 is the calibration curve of Pumilio obtained in Example 4;

FIG. 11 is the calibration curve of β-galactosidase obtained in Example5;

FIG. 12 is the calibration curve of β-galactosidase obtained in Example6;

FIG. 13 is the calibration curve of β-galactosidase obtained in Example7;

FIG. 14 is the calibration curve of β-galactosidase obtained in Example8;

FIG. 15 is the calibration curve of β-galactosidase obtained in Example9;

FIG. 16 is the calibration curve of Pumilio obtained in Example 10;

FIG. 17 is the calibration curve of Pumilio obtained in Example 11;

FIG. 18 is the calibration curve of Pumilio obtained in Example 12;

FIG. 19 is the calibration curve of Pumilio obtained in Example 13;

FIG. 20 is the calibration curve of Pumilio obtained in ReferenceExample 7;

FIG. 21 is the calibration curve of β-galactosidase obtained inReference Example 8;

FIG. 22 is the calibration curve of horseradish peroxidase obtained inExample 14; and

FIG. 23 is the calibration curve of Pumilio obtained in Example 15.

BEST MODE FOR CARRYING OUT THE INVENTION

[Novel Substrate]

The present invention relates to an androsterone derivative representedby the following formula (1).

(wherein, definitions of X, Y¹ and Y² are those of any one of (A), (B)and (C) described below.

(A) X represents a phosphate group, and Y¹ and Y² represent together amethylene group, or Y¹ represents hydrogen, and Y² represents a hydroxylgroup, a C₁₋₆ alkoxy group or a C₁₋₆ alkyl group,

(B) X represents one kind of a sugar moiety selected from a groupconsisting of glucose, galactose, fructose and mannose, Y¹ and Y²represent together a methylene group, or Y¹ represents hydrogen, and Y²represents hydrogen, a C₁₋₆ alkoxy group, or a C₁₋₆ alkyl group,

(C) X represents —O—CO—R, R represents a C₁₋₆ alkyl group or a phenylgroup, and Y¹ and Y² represent together a methylene group or an oxygenatom, or Y¹ represents hydrogen, and Y² represents hydrogen, a hydroxylgroup, a C₁₋₆ alkoxy group, or a C₁₋₆ alkyl group.)

The C₁₋₆ alkoxy group as an example of Y² may be, for example, a methoxygroup, an ethoxy group, a propoxy group and the like, and the C₁₋₆ alkylgroup may be, for example, a methyl group, an ethyl group, an iso-propylgroup, an n-propyl group, a tert-butyl group, an n-butyl group, ann-pentyl group, an n-hexyl group and the like.

The sugar of the sugar moiety can be comprised of glucose, galactose,fructose or mannose, and can be suitably selected depending on alabeling enzyme to be used.

—CO— in —O—CO—R means —(C═O)—, and the C₁₋₆ alkyl group in R may be, forexample, a methyl group, an ethyl group, an iso-propyl group, ann-propyl group, a tert-butyl group, an n-butyl group, an n-pentyl group,an n-hexyl group and the like. In addition, R may be a phenyl group, andmay have a functional group.

When Y¹ and Y² represent together an oxygen atom, X represents a sugarmoiety or —O—CO—R, specifically a sugar derivative of 5α-androsterone or5β-androsterone, for example, glucoside, galactoside, fructoside ormannoside. Meanwhile, glucoside, galactoside, fructoside and mannosideare formally referred to as glucopyranoside, galactopyranoside,fructopyranoside and mannopyranoside, respectively.

Specific examples of the compound of the present invention are listed inTable 1 to be described below. However, Table 1 includes compounds thatcan be used in the method and the kit of the present invention besidesthe specific examples of the compound of the present invention.

These compounds of the present invention can be synthesized based on orin reference to the methods described in Examples.

[Enzyme Cycling Assay]

The present invention relates to an enzyme assay using anantibody-enzyme complex, in which a quantification of a reaction productby the enzyme of the antibody-enzyme complex is performed by producingthio-NADH and/or thio-NADPH by enzyme cycling reaction using NADH and/orNADPH, thio-NAD and/or thio-NADP, and dehydrogenase (DH), and assayingthe amount of the produced thio-NADH and/or thio-NADPH, or measuring thechange of the color by the produced thio-NADH and/or thio-NADPH.

The antibody-enzyme complex consists of an antibody specific to a targetprotein antigen and an enzyme labeled with this antibody, and is used inthe assay of the above-mentioned target protein.

Furthermore, the present invention relates to a method of assaying anucleic acid probe using an enzyme-labeled nucleic acid probe, in whicha quantification of a reaction product by the enzyme of theenzyme-labeled nucleic acid probe is performed producing thio-NADHand/or thio-NADPH by enzymatic cycling reaction using NADH and/or NADPH,thio-NAD and/or thio-NADP, and dehydrogenase (DH), and assaying theamount of the produced thio-NADH and/or thio-NADPH, or measuring thechange of the color by the produced thio-NADH and/or thio-NADPH.

The enzyme-labeled nucleic acid probe consists of a nucleic acid probespecifically binding to a target nucleic acid and an enzyme labeled withthis nucleic acid probe, and is used in the assay of the above-mentionedtarget nucleic acid.

In the method of the present invention, the enzyme (labeling enzyme) ofthe antibody-enzyme complex or the enzyme-labeled nucleic acid probeused is alkaline phosphatase (ALP), galactosidase, glucosidase,fructosidase, mannosidase or peroxidase. As a substrate of the aboveenzyme, the androsterone derivative represented by the following formula(1) is used.

(i) In the case where the androsterone derivative represented by theformula (1) is used as a substrate of alkaline phosphatase, X representsa phosphate group, Y¹ and Y² represent together a methylene group, or Y¹represents hydrogen, and Y² represents hydrogen, a hydroxyl group, aC₁₋₆ alkoxy group, or a C₁₋₆ alkyl group. The androsterone derivativeencompassed in the case of (i) further includes compounds wherein Y² ishydrogen as well as the novel compounds of the present invention. Thecompound in which Y² is hydrogen is described in Tetrahedron, 1999, Vol.55, No. 17, p. 5465-5482. The entire contents of the description of thisliterature are hereby incorporated by reference.

(ii) In the case where the androsterone derivative represented by theformula (1) is used as a substrate of galactosidase, glucosidase,fructosidase or mannosidase, X represents a sugar moiety, the sugarmoiety represents one kind selected from the group consisting ofglucose, galactose, fructose and mannose, and Y¹ and Y² representtogether a methylene group or an oxygen atom, or Y¹ represents hydrogen,and Y² represents hydrogen, a hydroxyl group, a C₁₋₆ alkoxy group, or aC₁₋₆ alkyl group. The androsterone derivative encompassed in the case of(ii) further includes compounds in which Y¹ and Y² represent together anoxygen atom as well as the novel compounds of the present invention. Acompound in which X represents glucose when Y¹ and Y² represent togetheran oxygen atom, is described in Carbohydrate Research, 1971, Vol. 17,No. 1, p. 199-207. The entire contents of the description of thisliterature are hereby incorporated by reference. A compound in which Xrepresents glucose when Y² represents a hydroxyl group, is described inPhytochemistry, 1974, Vol. 13, No. 10, p. 2135-2142. The entire contentsof the description of this literature are hereby incorporated byreference.

(iii) In the case where the androsterone derivative represented by theformula (I) is used as a substrate of peroxidase, X represents —O—CO—R(provided that R represents a C₁₋₆ alkyl group or a phenyl group), Y¹and Y² represent together a methylene group or an oxygen atom, or Y¹represents hydrogen, and Y² represents hydrogen, a hydroxyl group, aC₁₋₆ alkoxy group, or a C₁₋₆ alkyl group. The androsterone derivativeencompassed in the case of (iii) is the novel compound of the presentinvention.

The ultra-high sensitivity assay of the present invention can be carriedout similarly to an ordinary enzyme immunoassay or nucleic acid probeassay. For example, a solid phase carrier may be used, which is used inan ordinary assay, for example, a solid phase carrier such as amicroplate or plastic tube, a magnetic bead and the like in which anantibody or nucleic acid probe specifically binding to a subject isimmobilized on the surface.

The antibody and the nucleic acid probe-enzyme complex can be preparedby an ordinary method.

The antibody constituting the antibody-enzyme complex can be suitablyselected from the antibodies that specifically bind to a subject to bemeasured by the enzyme immunoassay of the present invention. Forexample, the enzyme immunoassay of the present invention is used in anassay of a protein, and herein the antibody consisting of theantibody-enzyme complex is an antibody specifically binding to a proteinwhich is the subject. In addition, in this case, a basal plate is usedin which an antibody specifically binding to the subject protein isimmobilized on the surface. In addition, the antibody constituting theantibody-enzyme complex and the antibody immobilized on the basal platecan be a fragment of the antibody. In the present invention of theenzyme immunoassay, the subject is not limited to a protein, and can beany substance that is a measuring subject of an ordinary enzymeimmunoassay besides a protein.

In the nucleic acid probe enzyme complex, the probe can be suitablyselected similarly from the probes complementary to a nucleic acid probethat is complementary to a measuring subject.

In the method of the present invention, the quantification of a reactionproduct by the enzyme of the antibody-enzyme complex or the enzyme ofthe enzyme-labeled nucleic acid probe is performed by producingthio-NADH and/or thio-NADPH by enzymatic cycling reaction using NADHand/or NADPH, thio-NAD and/or thio-NADP, and dehydrogenase (DH), andassaying the amount of the produced thio-NADH and/or thio-NADPH, ormeasuring the change of the color by the produced thio-NADH and/orthio-NADPH.

In the method of the present invention, the concentration of eachcomponent can be in the range to be described below.

(1) Concentration range of the antibody-enzyme complex or theenzyme-labeled nucleic acid probe:

0.01 μg/ml to 1 mg/ml

(2) Concentration range of the substrate of the labeling enzyme:

1 μM to 500 mM

(3) Concentration range of the NADH and/or NADPH:

0.01 mM to 50 mM

(4) Concentration range of the thio-NAD and/or thio-NADP:

0.01 mM to 100 mM

(5) Concentration range of the dehydrogenase (DH):

0.01 U/ml to 5000 U/ml

The reaction conditions can be suitably determined by considering theoptimum temperature range of the labeling enzyme and the dehydrogenase(DH). For example, as for the reaction temperature, the reaction ispreferably carried out at room temperature from a point of simplemanipulation. However, the reaction can be carried out at highertemperature or lower temperature than room temperature by consideringthe optimum temperature range of the labeling enzyme and thedehydrogenase (DH).

The reaction time can be a time sufficient for accumulating thio-NADHand/or thio-NADPH, so as to enable the assay of the amount of theproduced thio-NADH and/or thio-NADPH, or the measurement of the changeof the color by the produced thio-NADH and/or thio-NADPH. However, theaccumulation amount of thio-NADH and/or thio-NADPH necessary for theassay or measurement varies depending on the conditions of the assay ormeasurement, and can be suitably determined depending on the conditions.

The cycling system using thio-NAD(P) in the enzymatic cycling system isa unique cycling system that appeared relatively recently. With thissystem, the cycling is performed that is, thio-NAD(P)/thio-NAD(P)H usingdehydrogenase (DH) with NAD(P)/NAD(P)H as a co-enzyme under conditionsof coexistence of NAD(P)/NAD(P)H and its analog, and the amplificationand the quantification are performed with thio-NAD(P)H (maximumabsorption wavelength: 400 nm, molar absorption coefficient=11,900) as asubstrate of dehydrogenase. The principle for measurement of thethio-NAD(P)cycling system is as described below.

Whereas the NADH exhibits the maximum absorption at 340 nm (molarabsorption coefficient=6,200), thio-NAD(P)H exhibits absorption in thevisible range (maximum absorption wavelength: 400 nm, molar absorptioncoefficient=11,900) and thus the thio-NAD(P)cycling method has anadvantage of enabling measurement using a popular absorptionspectrophotometer or a microplate reader for colorimetric measurement.

Using the advantages that measurement in the cycling system usingthio-NAD(P) can be performed by using a popular absorptionspectrophotometer or a microplate reader for colorimetric measurement,some conventional methods based on the increase of NADH absorption, suchas an assay of dehydrogenase activity and quantification of a substratethereof, have been improved to a method using thio-NAD. However, therehas been no report yet for use of this cycling system in sensitivityimprovement as a detection system such as an enzyme immunoassay.

In the present invention, the combination of a labeling enzyme and thecycling system enables the amplification reaction to be exponentialreaction for the first time, whereby resulting in sufficient sensitivityimprovement.

According to the assay of the present invention, as it is exemplified inthe enzyme immunoassay, the products produced with the enzyme complexand a substrate in combination are used as the substrate of the nextenzymatic cycling reaction, and the absorption of thio-NAD(P)H producedby the enzymatic cycling reaction is quantified colorimetrically. Theenzymatic cycling is performed with a dehydrogenase in this reaction,and thus either a reduced substrate or an oxidized substrate can be usedas a substrate of the enzymatic cycling reaction.

In the present invention, the labeling enzyme used as the enzymecomplex, the substrate thereof and the dehydrogenase subsequently usedin the cycling reaction have the properties as described below.

(1) The product of the labeling enzyme is androsterone or a derivativethereof;

(2) Commercially available and widely used labeling enzymes can be used;

(3) The turnover number of the enzymatic cycling is great; and

(4) Dehydrogenase used in this cycling reaction has no contamination ofthe labeling enzyme or similar activity to that of the labeling enzyme.

Examples of the dehydrogenase (DH) may include, for example,3α-hydroxysteroid dehydrogenase.

Examples of a representative combination of the labeling enzyme, thesubstrate and the dehydrogenase for the enzyme cycling that can be usedin the present invention will be described below. However, of course,the combination is not limited to these combinations.

TABLE 1 Labeled Enzyme Substrate Dehydrogenase α-glucosidase5α-androsterone 3α-glucoside 3α-hydroxysteroid dehydrogenaseα-glucosidase 5β-androsterone 3α-glucoside 3α-hydroxysteroiddehydrogenase α-glucosidase 3α-hydroxy-17β-methoxy-5α- 3α-hydroxysteroidandrostane 3-α-glucoside dehydrogenase α-glucosidase3α-hydroxy-17β-methoxy-5β- 3α-hydroxysteroid androstane 3-α-glucosidedehydrogenase β-glucosidase 5α-androsterone 3-β-glucoside3α-hydroxysteroid dehydrogenase β-glucosidase 5β-androsterone3-β-glucoside 3α-hydroxysteroid dehydrogenase β-glucosidase3α-hydroxy-17β-methoxy-5α- 3α-hydroxysteroid androstane 3-β-glucosidedehydrogenase β-glucosidase 3α-hydroxy-17β-methoxy-5β- 3α-hydroxysteroidandrostane 3-β-glucoside dehydrogenase β-galactosidase 5α-androsterone3-β-galacto- 3α-hydroxysteroid side dehydrogenase β-galactosidase5β-androsterone 3-β-galacto- 3α-hydroxysteroid side dehydrogenaseβ-galactosidase 3α-hydroxy-17β-methoxy-5α- 3α-hydroxysteroid androstane3-β galactoside dehydrogenase β-galactosidase 3α-hydroxy-17β-methoxy-5β-3α-hydroxysteroid androstane 3-β- galactoside dehydrogenase Acid phos-3α-hydroxy-17β-methyl-5α- 3α-hydroxysteroid phatase androstane3-phosphate dehydrogenase Acid phos- 3α-hydroxy-17β-methyl-5β-3α-hydroxysteroid phatase androstane 3-phosphate dehydrogenase Acidphos- 3α-hydroxy-17β-methoxy-5α- 3α-hydroxysteroid phatase androstane3-phosphate dehydrogenase Acid phos- 3α-hydroxy-17β-methoxy-5β-3α-hydroxysteroid phatase androstane 3-phosphate dehydrogenase Acidphos- 3α, 17α-dihydroxy-17β-methyl- 3α-hydroxysteroid phatase5α-androstane 3-phosphate dehydrogenase Acid phos-3α,17α-dihydroxy-17-methyl- 3α-hydroxysteroid phatase 5β-androstane3-phosphate dehydrogenase Acid phos- 3β, 17α-dihydroxy-17β-ethyl-3α-hydroxysteroid phatase 5α-androstane 3-phosphate dehydrogenase Acidphos- 3α, 17α-dihydroxy-17-ethyl- 3α-hydroxysteroid phatase5β-androstane 3-phosphate dehydrogenase Acid phos-17β-acetoxy-3α-hydroxy-5α- 3α-hydroxysteroid phatase androstane3-phosphate dehydrogenase Acid phos- 17β-acetoxy-3α-hydroxy-5β-3α-hydroxysteroid phatase androstane 3-phosphate dehydrogenase Alkalinephos- 3α-hydroxy-17β-methyl-5α- 3α-hydroxysteroid phatase androstane3-phosphate dehydrogenase Alkaline phos- 3α-hydroxy-17β-methyl-5β-3α-hydroxysteroid phatase androstane 3-phosphate dehydrogenase Alkalinephos- 3α-hydroxy-17β-methoxy-5α- 3α-hydroxysteroid phatase androstane3-phosphate dehydrogenase Alkaline phos- 3α-hydroxy-17β-methoxy-5β3α-hydroxysteroid phatase -androstane 3-phosphate dehydrogenase Alkalinephos- 3α,17α-dihydroxy-17β-methyl- 3α-hydroxysteroid phatase5α-androstane 3-phosphate dehydrogenase Alkaline phos- 3α,17α-dihydroxy-17β-methyl- 3α-hydroxysteroid phatase 5β-androstane3-phosphate dehydrogenase Alkaline phos- 3α, 17α-dihydroxy-17β-ethyl-3α-hydroxysteroid phatase 5α-androstane 3-phosphate dehydrogenaseAlkaline phos- 3α, 17α-dihydroxy-17β-ethyl- 3α-hydroxysteroid phatase5β-androstane 3-phosphate dehydrogenase Alkaline phos- 5β-androstane3-phosphate 3α-hydroxysteroid phatase dehydrogenase Alkaline phos-17β-acetoxy3α-hydroxy-5α- 3α-hydroxysteroid phatase androstane3-phosphate dehydrogenase Alkaline phos- 17β-acetoxy3α-hydroxy-5β-3α-hydroxysteroid phatase androstane 3-phosphate dehydrogenase Sulfatase5α-androstane 3-sulfate 3α-hydroxysteroid dehydrogenase Sulfatase5β-androstane 3-sulfate 3α-hydroxysteroid dehydrogenase Peroxidase3α-tert-butyl-peroxy-5α- 3α-hydroxysteroid androstane dehydrogenasePeroxidase 3α-tert-butyl-peroxy-5β- 3α-hydroxysteroid androstanedehydrogenase Peroxidase 3α-benzoyl-peroxy-5α- 3α-hydroxysteroidandrostane dehydrogenase Peroxidase 3α- benzoyl-peroxy-5β-3α-hydroxysteroid androstane dehydrogenase Peroxidase3α-acetyl-peroxy-5α-androstane 3α-hydroxysteroid dehydrogenasePeroxidase 3α- acetyl-peroxy-5β-androstane 3α-hydroxysteroiddehydrogenase

For example, when 3α-hydroxysteroid dehydrogenase is used as the cyclingenzyme among the combinations described above, examples of a candidatefor the substrate that can be used include androsterone,11β-hydroxyandrosterone, 11-oxoandrosterone and 11α-hydroxyandrosterone.

The substrate for the cycling is preferably those having fast enzymereaction rate (having high activity for the substrate) and reacting evenat a low concentration (having low Km value). In addition, a desirableproperty as the dehydrogenase is the reaction rate when usingthio-NAD(P) as a co-enzyme. In the dehydrogenation reaction ofandrosterone by other many dehydrogenases, the reaction rate whenthio-NAD is used as a co-enzyme is within several % of the reaction ratewhen NAD is used, while with 3α-hydroxysteroid dehydrogenase, thereaction rate with thio-NAD is about 59% of the reaction rate with NAD.Thus, 3α-hydroxysteroid dehydrogenase has a desirable property as thecycling enzyme of the present invention. Furthermore, widely usedalkaline phosphatase (ALP) can be used as the labeling enzyme to becombined, and the combination with alkaline phosphatase is preferablealso in a point of easy synthesis of a phosphate ester of anandrosterone derivative at the position 3 that is a substrate ofalkaline phosphatase.

In addition, similarly, glucosidase, which is widely used as a labelingenzyme, and glucoside of an androsterone derivative, and galactosidaseand galactoside of an androsterone derivative are a preferablecombination in a point of easy synthesis.

In addition, similarly, peroxidase, which is widely used as a labelingenzyme, and peroxide of an androsterone derivative, for example,tert-butyl peroxide are also a preferable combination in a point of easysynthesis.

A reaction example using androsterone 3-β-D galactoside as a substratewill be described below.

An enzyme immunoassay using alkaline phosphatase (ALP) as the enzyme ofthe antibody-enzyme complex will be explained below as an example.Alkaline phosphatase (ALP) is an enzyme widely used as a labelingenzyme, and when the androsterone derivative, the novel compound is usedas a substrate of ALP in an enzyme immunoassay using this ALP,androsterone is produced as a reaction product. Then, 3α-hydroxysteroiddehydrogenase (3α-HSD), which dehydrogenase uses the reaction product ofALP as a substrate, is used in the enzyme cycling reaction.

The thio-NAD cycling enables the amplification and the quantification ofthe substrate of dehydrogenase in an efficiency of hundreds of cyclesper minute if an appropriate dehydrogenase reaction is selected.Consequently, the activity of the enzyme presenting such a substrate asthe reaction product can be assayed by the thio-NAD cycling inultra-high sensitivity.

[Kit for Enzyme Cycling]

The present invention encompasses a kit for enzyme cycling methodincluding an enzyme labeled with a reactive carrier, a substratethereof, an enzyme for cycling reaction and thio-NAD and NADH as aco-enzyme thereof.

The reactive carrier represents an antibody, a nucleic acid probe,lectin and the like having a binding activity to a measuring subject.

The reactive carrier is not particularly limited if it is a materialsuitable for the measuring subject, or a material suitable for thelabeling enzyme and the substrate thereof.

More specifically, the present invention encompasses a kit for enzymecycling method including a labeling enzyme, a substrate thereof, anenzyme for cycling reaction and thio-NAD and NADH as a co-enzymethereof.

The kit of the present invention is a kit for enzyme immunoassayincluding reagents (1) to (5) below.

(1) alkaline phosphatase (ALP), glucosidase, galactosidase,fructosidase, mannosidase or peroxidase, which enzymes are labeled withan antibody specific to a target protein antigen,

(2) androsterone derivative represented by the formula (1), which is asubstrate of the enzyme,

(3) Dehydrogenase (DH),

(4) NADH and/or NADPH, and

(5) Thio-NAD and/or thio-NADP

Furthermore, the present invention is a kit for measuring a nucleic acidprobe, which includes reagents (1) to (5) below.

(1) alkaline phosphatase (ALP), glucosidase, galactosidase,fructosidase, mannosidase or peroxidase, which enzymes are labeled witha nucleic acid probe specifically binding to a target nucleic acid,

(2) androsterone derivative represented by the formula (1), which is asubstrate of the enzyme,

(3) Dehydrogenase (DH),

(4) NADH and/or NADPH, and

(5) Thio-NAD and/or thio-NADP

For the labeling enzyme, the dehydrogenase (DH) and the androsteronederivative represented by the formula (1), can be used are thosedescribed above in the method of the present invention as they are.

The kit of the present invention can be a commercial enzyme-labeledantibody and the like in combination with the reagents constituting thiskit. This kit can be used in an enzymatic immunoassay using enzymecycling method.

EXAMPLES

Hereinafter, this invention will be described based on Examples.However, such Examples can be modified variously in the technical levelof this field. Needless to say, this invention is not intended to belimited to these Examples only, in light of such technical level.

Hereinafter, the present invention will be more specifically describedin the following Examples.

Assay of alkaline phosphatase and Pumilio using thio-NAD cycling ofsystem of alkaline phosphatase (ALP) and androsterone 3-phosphate (A3P)as detection system.

Reference Example 1 Manufacture of ALP-labeled Antibody

A mouse-derived monoclonal antibody having antigen-specific reactivitywas dialysized three times with 0.1M citric acid buffer solution (pH3.5) for 30 minutes. The antibody solution was added with pepsin to 0.5%with respect to the amount of the antibody, stood at 37° C. for 1 hour,and then adjusted to neutral pH with 1.5 M Tris buffer solution (pH8.8). This mixed reaction solution was gel-filtered using a columnfilled with Superdex 200 (Amersham Biosciences, Inc.), to give F(ab′)₂.

ALP was labeled by ALP-labeling kit (DOJINDO LABORATORIES) using 100 μgof this F(ab′)₂.

Reference Example 2 Preparation of Insoluble Carrier Immobilized withAntibody 1

A solution of a guinea pig-derived polyclonal antibody havingantigen-specific reactivity dissolved in 50 mM sodium carbonate buffersolution (pH 9.6) in a concentration of 100 μg/ml, was added to eachwell of a flat-bottomed microplate (Nunc A/S) by 50 μl each, and stoodat room temperature for 1 hour, and then the antibody solution wascollected. 300 μl of TBS containing 2% bovine serum albumin (BSA) wasadded and stood at room temperature for 2 hours to perform the blockingtreatment, to give a polyclonal antibody-immobilized microplate.

Reference Example 3 Preparation of Insoluble Carrier Immobilized withAntibody 2

A solution of a guinea pig-derived polyclonal antibody havingantigen-specific reactivity dissolved in 50 mM sodium carbonate buffersolution (pH 9.6) in a concentration of 10 μg/ml was added to each wellof a flat-bottomed microplate (Nunc A/S) by 50 μl each, and stood atroom temperature for 1 hour. Then, the solution in the well was removedwith suction, 300 μl of TBS containing 0.1% bovine serum albumin (BSA)was added and stood at room temperature for 2 hours to perform theblocking treatment, and washed with TBS containing 0.05% Tween 20, togive a polyclonal antibody-immobilized microplate.

Comparative Example 1 Assay of Pumilio by One Step Method Using5α-androsterone 3-phosphate

Reaction Test Solution

0.2 M glycine buffer solution (pH 8.8)

1.5 mM thio-NAD

1 mM NADH

0.5 mM 5α-androsterone 3-phosphate

20 U/ml 3α-hydroxysteroid dehydrogenase

Sample for Assay

0.1, 0.5, 1, 2, 5, 10, 50, 100 and 200 ng/ml Pumilio

Method for Assay

To a microplate immobilized with the anti-Pumilio guinea pig polyclonalantibody prepared with the method of Reference Example 2, 50 μl of TBS(pH 7.5) containing 0.1% BSA solution containing purified Pumilio(standard substance) in a range of 0 to 200 ng/ml was added, and themicroplate was stirred at room temperature for 1 hour. Then, thesolution in the well was removed with suction, and then the microplatewas washed with TBS (pH 7.5) containing 0.05% Tween 20. The microplatewas added with 50 μl of TBS (pH 7.5) containing 0.1% BSA solutioncontaining the ALP-labeled anti-Pumilio mouse monoclonal antibodyprepared with the method of Reference Example 1 in a concentration of2.5 μg/ml, and the microplate was stirred for 1 hour. The solution inthe well was removed with suction, and then the microplate was washedwith TBS (pH 7.5) containing 0.05% Tween 20, and further washed withTBS. Then, 100 μl of the reaction test solution was added to each well,respectively, and the absorbance was measured for 30 minutes using a 405nm filter with a microplate reader (MTP-500 manufactured by CORONACORPORATION) while warming the microplate to 37° C. The amount of theabsorbance change for 30 minutes was plotted with respect to theconcentrations of the standard substance. The obtained straight linewell-dependent on the concentration is shown in FIG. 2.

Herein, poor reactivity of the enzyme (alkaline phosphatase) with thesubstrate (androsterone 3-phosphate), and inhibition of the substrate onthe cycling reaction came up as problems, and thus two-step method inwhich the enzyme reaction and the cycling reaction are split, was tried.In addition, it was found out that 3α-hydroxysteroid dehydrogenase, inthe cycling reaction an enzyme had phosphatase activity, and was a causefor the blank, and thus it was determined to use a buffer solutioncontaining phosphoric acid at the time of the cycling reaction of thetwo-step method.

Comparative Example 2 Assay of Pumilio by two-step method using5α-androsterone 3-phosphate

Reaction Test Solution A

0.1M Tris buffer solution (pH 8.3)

1 mM MgCl₂

0.1 mM 5α-androsterone 3-phosphate

Reaction Test Solution B

0.2 M disodium hydrogen phosphate-containing 0.2 M glycine buffersolution (pH 9.6)

3 mM thio-NAD

1 mM NADH

40 U/ml 3α-hydroxysteroid dehydrogenase

Sample for Assay

0.5, 0.75, 1, 2.5, 5, 7.5, 10, 12.5, 20, 50, 75, and 100 ng/ml Pumilio

Method for Assay

To a microplate immobilized with the anti-Pumilio guinea pig polyclonalantibody prepared in the method of Reference Example 3, 50 μl of TBS (pH7.5) containing 0.1% BSA solution containing purified Pumilio (standardsubstance) in a range of 0 to 100 ng/ml was added, and the microplatewas stirred at room temperature for 1 hour. Then, the solution in thewell was removed with suction, and then the microplate was washed withTBS (pH 7.5) containing 0.05% Tween 20. The microplate was added with 50μl of TBS (pH 7.5) containing 0.1% BSA solution containing theALP-labeled anti-Pumilio mouse monoclonal antibody prepared in themethod of Reference Example 1 in a concentration of 2.5 μg/ml, and themicroplate was stirred for 1 hour. The solution in the well was removedwith suction, and then the microplate was washed with TBS (pH 7.5)containing 0.05% Tween 20, and further washed with TBS. Then, 50 μl ofReaction test solution A was added to each well, respectively, and themicroplate was incubated at 37° C. for 30 minutes. Subsequently, 50 μlof Reaction test solution B was added to the well, and the absorbancewas measured for 30 minutes using a 405 nm filter with a microplatereader (MTP-500 manufactured by CORONA CORPORATION) while warming themicroplate to 37° C. The amount of the absorbance change for 30 minuteswas plotted with respect to the concentrations of the standardsubstance. The obtained straight line well-dependent on theconcentration is shown in FIG. 3.

Although the two-step method was tried, the sensitivity did not increaseas much as expected, and thus, it was determined to use 5β-androsterone3-phosphate, which has different structure from that of 5α-androsterone3-phosphate used so far, in order to improve the reactivity withalkaline phosphatase.

Comparative Example 3 Assay of Pumilio by One Step Method Using5β-androsterone 3-phosphate

Reaction Test Solution

0.1M Tris buffer solution (pH 8.6)

0.5 mM MgCl₂

1.5 mM thio-NAD

0.5 mM NADH

0.5 mM 5β-androsterone 3-phosphate

20 U/ml 3α-hydroxysteroid dehydrogenase

Sample for Assay

0.1, 0.5, 1, 2, 5, 8, 10, 20 and 50 ng/ml Pumilio

Method for Assay

To a microplate immobilized with the anti-Pumilio guinea pig polyclonalantibody prepared in the method of Reference Example 3, 50 μl of TBS (pH7.5) containing 0.1% BSA solution containing purified Pumilio (standardsubstance) in a range of 0 to 50 ng/ml was added, and the microplate wasstirred at room temperature for 1 hour. Then, the solution in the wellwas removed with suction, and then the microplate was washed with TBS(pH 7.5) containing 0.05% Tween 20. 50 μl of TBS (pH 7.5) containing0.1% BSA solution containing the ALP-labeled anti-Pumilio mousemonoclonal antibody prepared in the method of Reference Example 1 in aconcentration of 2.5 μg/ml was added, and the microplate was stirred for1 hour. The solution in the well was removed with suction, and then themicroplate was washed with TBS (pH 7.5) containing 0.05% Tween 20, andfurther washed with TBS. Then, to each well, 50 μl of the reaction testsolution was added, respectively, and the absorbance was measured for 30minutes using a 405 nm filter with a microplate reader (MTP-500manufactured by CORONA CORPORATION) while warming the microplate to 37°C. The amount of the absorbance change for 30 minutes was plotted withrespect to the concentrations of the standard substance. The obtainedstraight line well-dependent on the concentration is shown in FIG. 4.

Comparative Example 4 Assay of Pumilio by Two-step Method Using5β-androsterone 3-phosphate

Reaction Test Solution A

0.1M Tris buffer solution (pH 8.3)

1 mM MgCl₂

0.1 mM 5β-androsterone 3-phosphate

Reaction Test Solution B

0.2 M disodium hydrogen phosphate-containing 0.2 M glycine buffersolution (pH 9.6)

3 mM thio-NAD

1 mM NADH

40 U/ml 3α-hydroxysteroid dehydrogenase

Sample for Assay

0.005, 0.01, 0.05, 0.1, 0.25, 0.5, 0.75, 1, 2.5, 5, 10, 25 and 50 ng/mlPumilio

Method for Assay

To a microplate immobilized with the anti-Pumilio guinea pig polyclonalantibody prepared in the method of Reference Example 3, 50 μl of TBS (pH7.5) containing 0.1% BSA solution containing purified Pumilio (standardsubstance) in a range of 0 to 50 ng/ml was added, and the microplate wasstirred at room temperature for 1 hour. Then, the solution in the wellwas removed with suction, and then the microplate was washed with TBS(pH 7.5) containing 0.05% Tween 20. The microplate was added with 50 μlof TBS (pH 7.5) containing 0.1% BSA solution containing the ALP-labeledanti-Pumilio mouse monoclonal antibody prepared in the method ofReference Example 1 in a concentration of 2.5 μg/ml, and the microplatewas stirred for 1 hour. The solution in the well was removed withsuction, and then the microplate was washed with TBS (pH 7.5) containing0.05% Tween 20, and further washed with TBS. Then, 50 μl of Reactiontest solution A was added to each well, respectively, and the microplatewas incubated at 37° C. for 30 minutes. Subsequently, 50 μl of Reactiontest solution B was added to the well, and the absorbance was measuredfor 10 minutes using a 405 nm filter with a microplate reader (MTP-500manufactured by CORONA CORPORATION) while warming the microplate to 37°C. The amount of the absorbance change for 10 minutes was plotted withrespect to the concentrations of the standard substance. The obtainedstraight line well-dependent on the concentration is shown in FIG. 5.

It was found out that androsterone 3-phosphate was a substrate of thecycling reaction so far, and blank increase by this result became aproblem. It was ascertained that the cause was ketone at the position17, and thus it was determined to use a substrate in which the ketone atthe position 17 is removed or substituted. Synthesized3α-hydroxyandrostanes are shown in FIG. 6. In addition, the rotationnumbers of the cycling reactions using them as a substrate are shown inTable 2. Among them, the rotation number of 5α-androsterone was themaximum, and a substrate being close to this were3α-hydroxy-17β-methoxy-5β-androstane and 5α-dihydrotestosterone. Thus, aphosphate ester of the former, that is,3α-hydroxy-17β-methoxy-5β-androstane 3-phosphate was synthesized andapplied to the thio-NAD cycling.

TABLE 2 Rotation numbers of cycling reactions with 3α-hydroxyandrostanesCycle/min Relative value (%) 5α-Androsterone 623 100  5β-Androsterone384 62 5α-17-CH₃ 200 32 5α-17-deoxo 241 39 5α-17-CH₂ 308 49 5β-17-CH₃114 18 5β-17β-OCH₃ 458 74 5α-DHT 535 86 Glycochenodeoxycholate 129 21(Reference Example)

Example 1 Assay of Alkaline Phosphatase by One Step Method Using3α-hydroxy-17β-methoxy-5β-androstane 3-phosphate Synthesis of3α-hydroxy-17β-methoxy-5β-androstane 3-phosphate

To an anhydrous pyridine solution (4 mL) of phosphorus oxychloride (0.26mL), an anhydrous pyridine solution (4 mL) containing of3α-hydroxy-17β-methoxy-5β-androstane (200 mg) was dropped under icecooling, and the reaction solution was stirred further for 1 hour underice cooling. The reaction solution was poured into ice water, and thesolution was alkalified strongly (about pH 11), and then washed withether. Then, concentrated hydrochloric acid was added with the aqueouslayer to adjust pH to about 2, and then extracted with ether. The etherlayer was dried with anhydrous sodium sulfate, and then the solvent wasdistilled off under reduced pressure. The residue was recrystallizedwith tetrahydrofuran-hexane to give a colorless crystal (210 mg).

¹H-NMR (400 MHz, Methanol-d4) δ=0.73 (s, 3H, 18-CH₃), 0.95 (s, 3H,19-CH₃), 3.19 (1H, t, J=8.6 Hz, 17α-H), 3.32 (s, 3H, —OCH₃), 4.18 (m,1H, 3β-H)

ESI-HR-MS Calculated for C₂₀H₃₅O₅P—H: m/z 385.2149 [M-H]⁻. Found m/z385.2157 m.p. 187-188° C.

Reaction Test Solution

0.1M Tris buffer solution (pH 9.5)

0.2 mM MgCl₂

0.6 mM thio-NAD

4 mM NADH

0.5 mM 3α-hydroxy-17β-methoxy-5β-androstane 3-phosphate

20 U/ml 3α-hydroroxysteroid dehydrogenase

Sample for Assay

0.1, 0.2, 0.5, 1, and 2 mU/ml alkaline phosphatase (final concentration:0.05, 0.1, 0.25, 0.5, and 1 mU/ml)

Method for Assay

To a flat-bottomed microplate well, 50 μl of Tris buffer solution (pH9.5) containing the standard substance (alkaline phosphatase) in a rangeof 0 to 2 mU/ml was added, and subsequently, 50 μl of Reaction testsolution B was added to each well, respectively. Then, the absorbancewas measured for 60 minutes using a 405 nm filter with a microplatereader (MTP-500 manufactured by CORONA CORPORATION) while warming themicroplate to 37° C. The amount of the absorbance change for 60 minuteswas plotted with respect to the concentrations of the standardsubstance. The obtained straight line well-dependent on theconcentration is shown in FIG. 7.

Subsequently, which is an enzyme of the cycling reaction in respect ofinvestigation of the two-step method, as described above, a buffersolution containing phosphoric acid was used at the time of the cyclingreaction in order to inhibit the phosphatase activity in3α-hydroroxysteroid dehydrogenase. However, a buffer solution containingpyrophosphoric acid rather than the buffer solution containingphosphoric acid was adopted as a buffer solution further inhibiting thisphosphatase activity.

Example 2 Assay of Alkaline Phosphatase by Two-step Method Using3α-hydroxy-17β-methoxy-5β-androstane 3-phosphate

Reaction Test Solution A

0.1M Tris buffer solution (pH 9.5)

0.2 mM MgCl₂

1 mM 3α-hydroxy-17β-methoxy-5β-androstane 3-phosphate

Reaction Test Solution B

0.15 M pyrophosphate buffer solution (pH 9.5)

3 mM thio-NAD

1 mM NADH

40 U/ml 3α-hydroroxysteroid dehydrogenase

Sample for Assay

1, 5, 10, 15, 20 μU/ml alkaline phosphatase (final concentration: 0.5,2.5, 5, 7.5 and 10 μU/ml)

Method for Assay

To a flat-bottomed microplate well, 25 μl of Tris buffer solution (pH9.5) containing the standard substance (alkaline phosphatase) in a rangeof 0 to 20 μU/ml was added, and subsequently, 25 μl of Reaction testsolution A was added to the well, respectively. The reaction solutionwas mixed, and stood at 37° C. in an incubator for 1 hour. Then, 50 μlof Reaction test solution B was added to each well, respectively, andthe absorbance was measured for 20 minutes using a 405 nm filter with amicroplate reader (MTP-500 manufactured by CORONA CORPORATION) whilewarming the microplate to 37° C. The amount of the absorbance change for20 minutes was plotted with respect to the concentrations of thestandard substance. The obtained straight line well-dependent on theconcentration is shown in FIG. 8.

Reference Example 4

Purification of alkaline phosphatase (ALP)-labeled antibody ALP-labeledantibody was prepared using ALP-labeling-kit (DOJINDO LABORATORIES) from150 μg of F(ab′)₂ prepared in Reference Example 1. Since thisALP-labeled antibody solution contained various complexes having variousmolecular weight of ALP and Fab, and above solution had variation in theactivity, the prepared ALP-labeled antibody solution was gel-filteredusing superdex-200 column (Amersham Biosciences, Inc.) filled with Trisbuffer solution (pH 7.0) containing 1 mM MgCl₂, 0.1 mM ZnCl₂ and 0.01%BSA as an elution solution, and the activity of each fraction wascompared, and then only ALP-labeled antibody having great S/N ratio wascollected.

Reference Example 5

Preparation of Insoluble Carrier Immobilized with Antibody

A solution of a guinea pig-derived polyclonal antibody havingantigen-specific reactivity dissolved in 50 mM sodium carbonate buffersolution (pH 9.6) in a concentration of 10 μg/ml was added to each wellof a flat-bottomed microplate (Nunc A/S) by 50 μl each, and stood atroom temperature for 1 hour, and then the solution in the well wasremoved with suction, and washed with TBS (pH 7.5) containing of 0.5%Triton X-100. Then, 200 μl of TBS containing of 5% bovine serum albumin(BSA) was added and stood at 4° C. for 2 hours to overnight to performthe blocking treatment, and was washed with TBS (pH 7.5) containing of0.5% Triton X-100, to give a microplate immobilized with the polyclonalantibody.

Example 3 Assay of Pumilio by One Step Method

Reaction Test Solution Using 3α-hydroxy-17β-methoxy-5β-androstane3-phosphate

0.1M Tris buffer solution (pH 9.5)

1.5 mM thio-NAD

2.5 mM NADH

0.1 mM 3α-hydroxy-17β-methoxy-5β-androstane 3-phosphate

20 U/ml 3α-hydroroxysteroid dehydrogenase

Sample for Assay

0.1, 0.5, 2, 5, 10 and 25 ng/ml Pumilio

Method for Assay

To a microplate immobilized with the anti-Pumilio guinea pig polyclonalantibody prepared in the method of Reference Example 5, 50 μl of TBS (pH7.5) containing of 0.1% BSA solution containing purified Pumilio (thestandard substance) in a range of 0 to 25 ng/ml was added, and themicroplate was stirred at room temperature for 2 hours. Then, thesolution in the well was removed with suction, and then the well waswashed with TBS (pH 7.5) of containing 0.5% Triton X-100. 50 μl of TBS(pH 7.5) containing of 0.1% BSA solution containing the ALP-labeledanti-Pumilio mouse monoclonal antibody prepared in the method ofReference Example 4 in a concentration of approximately 0.8 μg/ml wasadded, and the microplate was stirred at 4° C. for 2 hours. The solutionin the well was removed with suction, and then the well was washed withTBS (pH 7.5) containing 0.5% Triton X-100, and further washed with TBS.Then, to each well, 50 μl of the reaction test solution was added,respectively, and the absorbance was measured for 60 minutes using a 405nm filter with a microplate reader (MTP-500 manufactured by CORONACORPORATION) while warming the microplate to 37° C. The amount of theabsorbance change for 60 minutes was plotted with respect to theconcentrations of the standard substance. The obtained straight linewell-dependent on the concentration is shown in FIG. 9.

Example 4 Assay of Pumilio by Two-step Method Using3α-hydroxy-17β-methoxy-5β-androstane 3-phosphate

Reaction Test Solution A

0.1M Tris buffer solution (pH 9.5)

0.1 mM MgCl₂

0.2 mM 3α-hydroxy-17β-methoxy-5β-androstane 3-phosphate

Reaction Test Solution B

0.15 M pyrophosphate buffer solution (pH 9.5)

3 mM thio-NAD

1 mM NADH

40 U/ml 3α-hydroroxysteroid dehydrogenase

Sample

0.01, 0.025, 0.05, 0.1, 0.25 and 0.5 ng/ml Pumilio

Method for Assay

To a microplate immobilized with the anti-Pumilio guinea pig polyclonalantibody prepared with the method of Reference Example 5, 50 μl of TBS(pH 7.5) containing 0.1% BSA solution containing purified Pumilio (thestandard substance) in a range of 0 to 0.5 ng/ml was added, and themicroplate was stirred at room temperature for 2 hours. Then, thesolution in the well was removed with suction, and then the well waswashed with TBS (pH 7.5) containing 0.5% Triton X-100. 50 μl of TBS (pH7.5) containing 0.1% BSA solution containing the ALP-labeledanti-Pumilio mouse monoclonal antibody prepared in the method ofReference Example 4 in approximately 0.2 μg/ml and the rabbit F(ab′)₂ ina concentration of 1 mg/ml was added, and the microplate was stirred at4° C. for 2 hours. The solution in the well was removed with suction,and then the residue was washed with TBS (pH 7.5) containing 0.5% TritonX-100, and further washed with TBS. Then, 50 μl of Reaction testsolution A was added to each well, respectively, and the microplate wasincubated at 37° C. for 30 minutes. Subsequently, 50 μl of Reaction testsolution B was added to the well, and the absorbance was measured for 30minutes using a 405 nm filter with a microplate reader (MTP-500manufactured by CORONA CORPORATION) while warming the microplate to 37°C. The amount of the absorbance change for 30 minutes was plotted withrespect to the concentrations of the standard substance. The obtainedstraight line well-dependent on the concentration is shown in FIG. 10.

Although a substrate modified at the position 17 was used, the blankreaction could not be completely removed as described above. Thus,β-galactosidase was used as a labeling enzyme of ELISA, and androsteroneβ-D-galactoside was synthesized as a substrate thereof, and theinvestigation proceeded.

Assay of β-galactosidase and Pumilio thio-NAD cycling of system ofβ-galactosidase and androsterone 3-β-D-galactoside using as detectionsystem.

Example 5 Assay of β-galactosidase by Two-step Method Using5β-androsterone β-D-galactoside Synthesis of 5β-androsterone3-β-D-galactoside (Formal Name: 5β-androsterone3-(3-D-galactopyranoside)

To a dichloromethane solution (15 mL) of 5β-androsterone (290 mg), MS4A(1.25 g) and phenyl 2,3,4,6-O-tetrabenzoyl-1-thio-β-D-garactopyranoside(1.03 g) were added and the reaction solution was stirred at −20° C. for15 minutes. Then, the reaction solution was added with N-iodosuccinimide(435 mg) and trifluoromethane sulfonic acid (15 mg), stirred at −20° C.for 2 hours, and then reacted further at room temperature for 2 hours.The reaction solution was added with 250 μL of triethylamine to stop thereaction, and washed with a mixed solution of saturated sodium carbonateand thiosodium sulfate in 3:1, and saturated saline sequentially. Theorganic layer was dried with anhydrous sodium sulfate, and then thesolvent was distilled off under reduced pressure. The residue wasdissolved in methanol (15 mL), and added with 28% sodium methoxide (500μL) and the reaction solution was stirred for 2 hours. The reactionsolution was added with acetic acid to be neutralized, and theninspissated under reduced pressure. The residue was purified with silicagel column chromatography (24×240 mm, 10% methanol-chloroform), and thenrecrystallized with methanol to give a colorless crystal (360 mg).

¹H-NMR (400 MHz, Methanol-d4) δ=0.85 (s, 3H, 18-CH₃), 0.97 (s, 3H,19-CH₃), 3.44-3.49 (bm, 3H, H-2′, H-3′, H-5′), 3.70 (d, 1H, H-6′a), 3.72(d, 1H, H-6′b), 3.73 (m, 1H, 3β-H), 3.81 (d, 1H, H-4′), 4.33 (d, 1H,H-1′, J_(1′,2′)=6.2 Hz)

ESI-HR-MS Calculated for C₂₅H₄₀O₇—H: m/z 451.2701 [M-H]⁻. Found m/z451.2711 m.p. 130-135° C.

Reaction Test Solution A

0.1M phosphate buffer solution (pH 7.3)

0.2 mM MgCl₂

0.1 mM 5β-androsterone β-D-galactoside

Reaction Test Solution B

0.1M pyrophosphate buffer solution (pH 9.0)

1.2 mM thio-NAD

3.6 mM NADH

40 U/ml 3α-hydroroxysteroid dehydrogenase

Sample

1, 4, 10, 40, 100, and 200 pg/ml β-galactosidase (final concentration:0.5, 2, 5, 20, 50 and 100 pg/ml)

Method for Assay

To a flat-bottomed microplate well, 25 μl of a phosphate buffer solution(pH 7.3) containing the standard substance (β-galactosidase) in a rangeof 0 to 200 pg/ml was added, and subsequently, 25 μl of Reaction testsolution A was added to the well, respectively. The reaction solutionwas mixed, and the microplate was incubated at 37° C. for 60 minutes.Then, 50 μl of Reaction test solution B was added to each well,respectively, and the absorbance was measured for 30 minutes using a 405nm filter with a microplate reader (MTP-500 manufactured by CORONACORPORATION) while warming the microplate to 37° C. The amount of theabsorbance change for 30 minutes was plotted with respect to theconcentrations of the standard substance. The obtained straight linewell-dependent on the concentration is shown in FIG. 11.

Example 6 Assay of β-galactosidase by One Step Method Using5β-androsterone β-D-galactoside

Reaction Test Solution

0.1M phosphate buffer solution (pH 7.7)

10 mM MgCl₂

6 mM thio-NAD

1 mM NADH

0.1 mM 5β-androsterone β-D-galactoside

40 U/ml 3α-hydroroxysteroid dehydrogenase

Sample

5, 10, 25, 50, 100, and 250 pg/ml β-galactosidase (final concentration:2.5, 5, 12.5, 25, 50 and 125 pg/ml)

Method for Assay

To a flat-bottomed microplate well, 50 μl of a phosphate buffer solution(pH 7.7) containing the standard substance (β-galactosidase) in a rangeof 0 to 250 pg/ml was added, and subsequently, 50 μl of Reaction testsolution B was added to each well, respectively. The absorbance wasmeasured for 120 minutes using a 405 nm filter with a microplate reader(MTP-500 manufactured by CORONA CORPORATION) while warming themicroplate to 37° C. The amount of the absorbance change for 120 minuteswas plotted with respect to the concentrations of the standardsubstance. The obtained straight line well-dependent on theconcentration is shown in FIG. 12.

Example 7 Assay of β-galactosidase by Two-step Method Using5β-androsterone β-D-galactoside

Reaction Test Solution A

0.1M phosphate buffer solution (pH 7.5)

10 mM MgCl₂

0.1 mM 5β-androsterone β-D-galactoside

Reaction Test Solution B

0.1M pyrophosphate buffer solution (pH 9.0)

4.8 mM thio-NAD

0.4 mM NADH

40 U/ml 3α-hydroroxysteroid dehydrogenase

Sample

1, 4, 10, 40, 100 and 200 pg/ml β-galactosidase (final concentration:0.5, 2, 5, 20, 50 and 100 pg/ml)

Method for Assay

To a flat-bottomed microplate well, 25 μl of a phosphate buffer solution(pH 7.5) containing the standard substance (β-galactosidase) in a rangeof 0 to 200 pg/ml was added, and subsequently, 25 μl of Reaction testsolution A was added to the well, respectively. The reaction solutionwas mixed, and the microplate was incubated at 37° C. for 60 minutes.Then, 50 μl of Reaction test solution B was added to each well,respectively, and the absorbance was measured for 30 minutes using a 405nm filter with a microplate reader (MTP-500 manufactured by CORONACORPORATION) while warming the microplate to 37° C. The amount of theabsorbance change for 30 minutes was plotted with respect to theconcentrations of the standard substance. The obtained straight linewell-dependent on the concentration is shown in FIG. 13.

Example 8 Assay of β-galactosidase by One Step Method Using3α-hydroxy-17β-methoxy-5β-androstane β-D-galactoside Synthesis of3α-hydroxy-17β-methoxy-5β-androstane β-D-galactoside (Formal Name:3α-hydroxy-17β-methoxy-5β-androstane 3-β-D-galactopyranoside)

To a dichloromethane solution (15 mL) of3α-hydroxy-17β-methoxy-5β-androstane (306 mg), MS4A (1.25 g) and phenyl2,3,4,6-O-tetrabenzoyl-1-thio-β-D-garactopyranoside (1.03 g) were addedand the reaction solution was stirred at −20° C. for 15 minutes. Then,N-iodosuccinimide (435 mg) and trifluoromethane sulfonic acid (15 mg)were added the reaction solution was stirred at −20° C. for 2 hours, andthen reacted further at room temperature for 2 hours. The reactionsolution was added with 250 μL triethylamine to stop the reaction, andsequentially washed with a mixed solution of saturated sodium carbonateand thiosodium sulfate in 3:1 and saturated saline. The organic layerwas dried with anhydrous sodium sulfate, and then the solvent wasdistilled off under reduced pressure. The residue was dissolved inmethanol (15 mL) and added with 28% sodium methoxide (500 μL) and thereaction solution was stirred for 2 hours. The reaction solution wasadded with acetic acid to be neutralized, and then inspissated underreduced pressure. The residue was purified with silica gel columnchromatography (24×240 mm, 10% methanol-chloroform), and thenrecrystallized with acetone-hexane to give a colorless crystal (383 mg).

¹H-NMR (400 MHz, Methanol-d4) δ=0.73 (s, 3H, 18-CH₃), 0.95 (s, 3H,19-CH₃), 3.26 (t, 1H, J=8.6 Hz, 17α-H), 3.32 (s, 3H, —OCH₃), 3.45-3.51(bm, 3H, H-2′, H-3′, H-5′), 3.71 (s, 1H, H-6′a), 3.73 (d, 1H, H-6′b),3.74 (m, 1H, 3β-H), 3.81 (d, 1H, H-4′), 4.33 (d, 1H, H-1′, J_(1′,2′)=7.5Hz)

ESI-HR-MS Calculated for C₂₆H₄₄O₇—H: m/z 467.3014 [M-H]⁻. Found m/z451.3026 m.p. 197-199° C.

Reaction Test Solution

0.1M phosphate buffer solution (pH 8.5)

10 mM MgCl₂

6 mM thio-NAD

1 mM NADH

0.2 mM 3α-hydroxy-17β-methoxy-5β-androstane β-D-galactoside

20 U/ml 3α-hydroroxysteroid dehydrogenase

Sample

5, 10, 25, 50, 100, and 250 pg/ml β-galactosidase (final concentration:2.5, 5, 12.5, 25, 50 and 125 pg/ml)

Method for Assay

To a flat-bottomed microplate well, 50 μl of a phosphate buffer solution(pH 8.5) containing the standard substance (β-galactosidase) in a rangeof 0 to 250 pg/ml was added, and subsequently, 50 μl of Reaction testsolution B was added to each well, respectively, and the absorbance wasmeasured for 120 minutes using a 405 nm filter with a microplate reader(MTP-500 manufactured by CORONA CORPORATION) while warming themicroplate to 37° C. The amount of the absorbance change for 120 minuteswas plotted with respect to the concentrations of the standardsubstance. The obtained straight line well-dependent on theconcentration is shown in FIG. 14.

Example 9 Assay of β-galactosidase by Two-step Method Using3α-hydroxy-17β-methoxy-5β-androstane β-D-galactoside

Reaction Test Solution A

0.1M phosphate buffer solution (pH 7.5)

4 mM MgCl₂

0.1 mM 3α-hydroxy-17β-methoxy-5β-androstane β-D-galactoside

Reaction Test Solution B

0.1M pyrophosphate buffer solution (pH 9.8)

1.2 mM thio-NAD

1 mM NADH

40 U/ml 3α-hydroroxysteroid dehydrogenase

Sample

1, 4, 10, 40, 100 and 200 pg/ml β-galactosidase (final concentration:0.5, 2, 5, 20, 50 and 100 pg/ml)

Method for Assay

To a flat-bottomed microplate well, 25 μl of a phosphate buffer solution(pH 7.5) containing the standard substance (β-galactosidase) in a rangeof 0 to 200 pg/ml was added, and subsequently, 25 μl of Reaction testsolution A was added to the well, respectively. The reaction solutionwas mixed, and the microplate was incubated at 37° C. for 60 minutes.Then, 50 μl of Reaction test solution B was added to each well,respectively, and the absorbance was measured for 30 minutes using a 405nm filter with a microplate reader (MTP-500 manufactured by CORONACORPORATION) while warming the microplate to 37° C. The amount of theabsorbance change for 30 minutes was plotted with respect to theconcentrations of the standard substance. The obtained straight linewell-dependent on the concentration is shown in FIG. 15.

Examples of measurements of Pumilio using androsterone β-D-galactosideare described below. However, there is no antibody that hasantigen-specific reactivity and is directly labeled withβ-galactosidase, and thus Labeled StreptAvidin-Biotin (LSAB) method istried.

Reference Example 6 Manufacture of Biotin-labeled Antibody

A biotin-labeled antibody was prepared from a biotin-labeling-kit(DOJINDO LABORATORIES) using 50 μg of F(ab′)₂ obtained in ReferenceExample 1.

Example 10 Assay of Pumilio by One Step Method Using5β-androsteroneβ-D-galactoside

Reaction Test Solution

0.1M phosphate buffer solution (pH 7.7)

5 mM MgCl₂

1.2 mM thio-NAD

0.8 mM NADH

0.05 mM 5β-androstaneβ-D-galactoside

20 U/ml 3α-hydroroxysteroid dehydrogenase

Sample

0.05, 0.1, 0.25, 0.5, 1 and 2.5 ng/ml Pumilio

Method for Assay

To a microplate immobilized with the anti-Pumilio guinea pig polyclonalantibody prepared with the method of Reference Example 5, 50 μl of TBS(pH 7.5) containing 0.1% BSA solution containing purified Pumilio (thestandard substance) in a range of 0 to 2.5 ng/ml was added, and themicroplate was stirred at room temperature for 2 hours. Then, thesolution in the well was removed with suction, and then the well waswashed with TBS (pH 7.5) containing 0.5% Triton X-100, and added with 50μl of TBS (pH 7.5) containing 0.1% BSA solution containing thebiotin-labeled anti-Pumilio mouse monoclonal antibody prepared in themethod of Reference Example 6 in a concentration of approximately 0.1μg/ml, and the microplate was stirred at room temperature for 1 hour.The solution in the well was removed with suction, and then the well waswashed with TBS (pH 7.5) containing 0.5% Triton X-100, and added with 50μl of TBS (pH 7.5) containing 0.1% BSA solution containingβ-galactosidase-labeled streptavidin (F. Hoffmann-La Roche Ltd) in aconcentration of 0.1 U conjugate/ml, and stirred at room temperature for30 minutes. The solution in the well was removed with suction, and thenthe well was washed with TBS (pH 7.5) containing 0.5% Triton X-100, andfurther washed with TBS. Then, to each well, 50 μl of the reaction testsolution was added, respectively, and the absorbance was measured for 60minutes using a 405 nm filter with a microplate reader (MTP-500manufactured by CORONA CORPORATION) while warming the microplate to 37°C. The amount of the absorbance change for 60 minutes was plotted withrespect to the concentrations of the standard substance. The obtainedstraight line well-dependent on the concentration is shown in FIG. 16.

Example 11 Assay of Pumilio by Two-step Method Using 5β-androsteroneβ-D-galactoside

Reaction Test Solution A

0.1M phosphate buffer solution (pH 7.5)

0.1 mM MgCl₂

0.05 mM 5β-androsterone β-D-galactoside

Reaction Test Solution B

0.1M pyrophosphate buffer solution (pH 9.0)

3 mM thio-NAD

2 mM NADH

40 U/ml 3α-hydroroxysteroid dehydrogenase

Sample

0.05, 0.1, 0.5, 1, 2.5 and 5 ng/ml Pumilio

Method for Assay

To a microplate immobilized with the anti-Pumilio guinea pig polyclonalantibody prepared with the method of Reference Example 5, 50 μl of TBS(pH 7.5) containing 0.1% BSA solution containing purified Pumilio (thestandard substance) in a range of 0 to 5 ng/ml was added, and themicroplate was stirred at room temperature for 2 hours. Then, thesolution in the well was removed with suction, and then the well waswashed with TBS (pH 7.5) containing 0.5% Triton X-100, and added with 50μl of TBS (pH 7.5) containing 0.1% BSA solution containing thebiotin-labeled anti-Pumilio mouse monoclonal antibody prepared with themethod of Reference Example 6 in a concentration of approximately 0.1μg/ml, and the microplate was stirred at room temperature for 1 hour.The solution in the well was removed with suction, and then the well waswashed with TBS (pH 7.5) containing 0.5% Triton X-100, and added with 50μl of TBS (pH 7.5) containing 0.1% BSA solution containingβ-galactosidase-labeled streptavidin (F. Hoffmann-La Roche Ltd) in aconcentration of 0.1 U conjugate/ml, and stirred at room temperature for30 minutes. The solution in the well was removed with suction, and thenthe well was washed with TBS (pH 7.5) containing 0.5% Triton X-100, andfurther washed with TBS. Then, to each well, 50 μl of Reaction testsolution A was added, respectively, and the microplate was incubated at37° C. for 30 minutes. Subsequently, 50 μl of Reaction test solution Bwas added to the well, and the absorbance was measured for 30 minutesusing a 405 nm filter with a microplate reader (MTP-500 manufactured byCORONA CORPORATION) while warming the microplate to 37° C. The amount ofthe absorbance change for 30 minutes was plotted with respect to theconcentrations of the standard substance. The obtained straight linewell-dependent on the concentration is shown in FIG. 17.

Example 12 Assay of Pumilio by One Step Method Using3α-hydroxy-17β-methoxy-5β-androstane β-D-galactoside

Reaction Test Solution

0.1M phosphate buffer solution (pH 8.5)

1.2 mM thio-NAD

0.8 mM NADH

0.05 mM 3α-hydroxy-17β-methoxy-5β-androstaneβ-D-galactoside

20 U/ml 3α-hydroroxysteroid dehydrogenase

Sample

0.1, 0.5, 1, 2.5, 5 and 10 ng/ml Pumilio

Method for Assay

To a microplate immobilized with the anti-Pumilio guinea pig polyclonalantibody prepared with the method of Reference Example 5, 50 μl of TBS(pH 7.5) containing 0.1% BSA solution containing purified Pumilio (thestandard substance) in a range of 0 to 10 ng/ml was added, and themicroplate was stirred at room temperature for 2 hours. Then, thesolution in the well was removed with suction, and then the well waswashed with TBS (pH 7.5) containing 0.5% Triton X-100, and added with 50μl of TBS (pH 7.5) containing 0.1% BSA solution containing thebiotin-labeled anti-Pumilio mouse monoclonal antibody prepared in themethod of Reference Example 6 in a concentration of approximately 0.1μg/ml, and the microplate was stirred at room temperature for 1 hour.The solution in the well was removed with suction, and then the well waswashed with TBS (pH 7.5) containing 0.5% Triton X-100, and added with 50μl of TBS (pH 7.5) containing 0.1% BSA solution containingβ-galactosidase-labeled streptavidin (F. Hoffmann-La Roche Ltd) in aconcentration of 0.1 U conjugate/ml, and stirred at room temperature for30 minutes. The solution in the well was removed with suction, and thenthe well was washed with TBS (pH 7.5) containing 0.5% Triton X-100, andfurther washed with TBS. Then, to each well, 50 μl of the reaction testsolution was added, respectively, and the absorbance was measured for 60minutes using a 405 nm filter with a microplate reader (MTP-500manufactured by CORONA CORPORATION) while warming the microplate to 37°C. The amount of the absorbance change for 60 minutes was plotted withrespect to the concentrations of the standard substance. The obtainedstraight line well-dependent on the concentration is shown in FIG. 18.

Example 13 Assay of Pumilio by Two-Step Method Using3α-hydroxy-17β-methoxy-5β-androstaneβ-D-galactoside

Reaction Test Solution A

0.1M phosphate buffer solution (pH 7.0)

0.05 mM 3α-hydroxy-17β-methoxy-5β-androstaneβ-D-galactoside

Reaction Test Solution B

0.1M pyrophosphate buffer solution (pH 9.0)

2.4 mM thio-NAD

2 mM NADH

40 U/ml 3α-hydroroxysteroid dehydrogenase

Sample

0.25, 0.5, 1, 2.5, 5, 10 ng/ml Pumilio

Method for Assay

To a microplate immobilized with the anti-Pumilio guinea pig polyclonalantibody prepared with the method of Reference Example 5, 50 μl of TBS(pH 7.5) containing 0.1% BSA solution containing purified Pumilio (thestandard substance) in a range of 0 to 10 ng/ml was added, and themicroplate was stirred at room temperature for 2 hours. Then, thesolution in the well was removed with suction, and then the well waswashed with TBS (pH 7.5) containing 0.5% Triton X-100, and 50 μl of TBS(pH 7.5) containing 0.1% BSA solution containing the biotin-labeledanti-Pumilio mouse monoclonal antibody prepared with the method ofReference Example 6 in a concentration of approximately 0.3 μg/ml wasadded, and the microplate was stirred at room temperature for 1 hour.The solution in the well was removed with suction, and then the well waswashed with TBS (pH 7.5) containing 0.5% Triton X-100, and added with 50μl of TBS (pH 7.5) containing 0.1% BSA solution containingβ-galactosidase-labeled streptavidin (F. Hoffmann-La Roche Ltd) in aconcentration of 0.1 U conjugate/ml, and stirred at room temperature for30 minutes. The solution in the well was removed with suction, and thenthe well was washed with TBS (pH 7.5) containing 0.5% Triton X-100, andfurther washed with TBS. Then, 50 μl of Reaction test solution A wasadded to each well, respectively, and the microplate was incubated at37° C. for 60 minutes. Subsequently, to the well, 50 μl of Reaction testsolution B was added, and the absorbance was measured for 30 minutesusing a 405 nm filter with a microplate reader (MTP-500 manufactured byCORONA CORPORATION) while warming the microplate to 37° C. The amount ofthe absorbance change for 30 minutes was plotted with respect to theconcentrations of the standard substance. The obtained straight linewell-dependent on the concentration is shown in FIG. 19.

Reference Example 7 Assay of Pumilio Using p-nitrophenyl phosphate(p-NPP)

Reaction Test Solution

0.1M glycine buffer solution (pH 10.3)

1 mM MgCl₂

1 mM ZnCl₂

1 mg/ml p-NPP

Sample for Assay

0, 1, 2.5, 5, 10 and 25 ng/ml Pumilio

Method for Assay

To a microplate immobilized with the anti-Pumilio guinea pig polyclonalantibody prepared with the method of Reference Example 3, 50 μl of TBS(pH 7.5) containing 0.1% BSA solution containing purified Pumilio (thestandard substance) in a range of 0 to 25 ng/ml was added, and themicroplate was stirred at room temperature for 1 hour. Then, thesolution in the well was removed with suction, and then the microplatewas washed with TBS (pH 7.5) containing 0.05% Tween 20. 50 μl of TBS (pH7.5) containing 0.1% BSA solution containing the ALP-labeledanti-Pumilio mouse monoclonal antibody prepared with the method ofReference Example in a concentration of 1 μg/ml was added, and themicroplate was stirred for 1 hour. The solution in the well was removedwith suction, and then the microplate was washed with TBS (pH 7.5)containing 0.05% Tween 20. 100 μl of the reaction test solution wasadded to each well, and the absorbance was measured for 30 minutes usinga 405 nm filter with a microplate reader (MTP-500 manufactured by CORONACORPORATION) while warming the microplate to 37° C. The amount of theabsorbance change for 30 minutes was plotted with respect to theconcentrations of the standard substance. The obtained straight linewell-dependent on the concentration is shown in FIG. 20.

Reference Example 8 Assay of β-galactosidase Using o-nitrophenylβ-D-galactopyranoside (ONPG)

Reaction Test Solution

0.1M phosphate buffer solution (pH 7.3)

0.2 mM 2-mercaptoethanol

1 mM MgCl₂

10 mM ONPG

Sample for Assay

2, 10, 20 and 50 ng/ml β-galactosidase (final concentration: 1, 5, 10and 25 ng/ml)

Method for Assay

To a flat-bottomed microplate well, 50 μl of a phosphate buffer solution(pH 7.3) containing the standard substance (β-galactosidase) in a rangeof 0 to 25 ng/ml was added, and subsequently, to the well, 50 μl of thereaction test solution was added, respectively, and the absorbance wasmeasured for 60 minutes using a 415 nm filter with a microplate reader(MTP-500 manufactured by CORONA CORPORATION) while warming themicroplate to 37° C. The amount of the absorbance change for 60 minuteswas plotted with respect to the concentrations of the standardsubstance. The obtained straight line well-dependent on theconcentration is shown in FIG. 21.

Calculation Method of Detection Limit and Quantification Limit

The amount of the absorbance change of each measurement result isplotted with respect to the concentrations of the standard substance,and only the concentrations in parallel on the straight line areextracted to prepare a fitted curve. Then, the standard deviation of theblank (not including the standard substance; n≧3) is calculated, and the3-fold value and 10-fold value thereof are divided by the slope of thefitted curve, and the obtained numerical values are taken as thedetection limit and the quantification limit, respectively.

The detection limit and the quantification limit calculated from eachexperiment result are summarized in Tables 3, 4 and 5.

TABLE 3 Detection limit and quantification limit of alkaline phosphataseand β-Galactosidase ALP and β-Gal Assay (unit: mol) DetectionQuantification Enzyme Substrate Method limit limit ALP 17-OMe 5β-A3POne-step 3.25 × 10⁻¹⁸ 1.08 × 10⁻¹⁷ Two-steps 8.19 × 10⁻²⁰ 2.73 × 10⁻¹⁹β-Gal 5α-AG Two-steps 3.89 × 10⁻¹⁹  1.3 × 10⁻¹⁸ 5β-AG One-step 3.31 ×10⁻¹⁹  1.1 × 10⁻¹⁸ Two-steps 7.48 × 10⁻²⁰ 2.49 × 10⁻¹⁹ 17-OMe 5β-AGOne-step 3.21 × 10⁻¹⁹ 1.07 × 10⁻¹⁸ Two-steps 1.09 × 10⁻¹⁹ 3.64 × 10⁻¹⁹ONPG  4.5 × 10⁻¹⁷  1.5 × 10⁻¹⁶ 4-MUG 9.76 × 10⁻¹⁹ 3.25 × 10⁻¹⁷

TABLE 4 Detection limit and quantification limit of Pumilio protein(β-Galactosidase system) ELISA Assay (unit: mol) QuantificationDetection limit limit 5β-AG One-step 2.21 × 10⁻¹⁷ 7.35 × 10⁻¹⁷ Two-steps1.65 × 10⁻¹⁷ 5.51 × 10⁻¹⁷ 17-OMe 5β-AG One-step 3.24 × 10⁻¹⁷ 1.08 ×10⁻¹⁶ Two-steps 3.17 × 10⁻¹⁷ 1.06 × 10⁻¹⁶ 4-MUG 2.07 × 10⁻¹⁶ 6.91 ×10⁻¹⁶

TABLE 5 Detection limit and quantification limit of Pumilio protein(Alkaline phosphatase system) ELISA Assay Quantification SubstrateDetection limit limit 5α-A3P One-step 3.57 x 10⁻¹⁴  7.3 x 10⁻¹⁴Two-steps 2.63 x 10⁻¹⁶ 8.76 x 10⁻¹⁶ 5β-A3P One-step 3.96 x 10⁻¹⁵ 1.32 x10⁻¹⁴ Two-steps  1.2 x 10⁻¹⁶  4 x 10⁻¹⁶ 17-OMe 5β- One-step  4.0 x 10⁻¹⁷ 1.3 x 10⁻¹⁶ A3P Two-steps 2.45 x 10⁻¹⁸ 8.17 x 10⁻¹⁸ p-NPP 3.97 x 10⁻¹⁶1.32 x 10⁻¹⁵

Example 14 Assay of Horseradish Peroxidase by One Step Method Using3α-tert-butyl-peroxy-5β-androsterone

Synthesis of 3α-tert-butyl-peroxy-5β-androsterone

3α-chloro-5β-androsterone (100 mg) was dissolved in a dichloromethanesolution (15 mL), to prepare the reaction test solution. To the presentsolution, a solution of tert-butyl-hydroperoxide (30 mg) dissolved in anaqueous solution (15 mL) of 20% sodium hydroxide was added, and thereaction solution was reacted with stirring at room temperature for 2hours. The organic layer was collected from the reaction solution, andwashed with an aqueous solution of saturated sodium hydrogen carbonateand saturated saline, sequentially. The organic layer was dehydratedwith anhydrous sodium sulfate, and then the solvent was distilled offunder reduced pressure. The residue was purified with silica gelchromatography (24×240 mm, 10% methanol-chloroform), and then 88 mg ofthe present compound was obtained. Identification of the presentcompound was performed from ¹H-NMR, ESI-HR-MS and m.p. (melting point).

Reaction Test Solution

0.1M phosphate buffer solution (pH 7.0)

10 mM MgCl₂

6 mM thio-NAD

1 mM NADH

0.1 mM 3α-tert-butyl-peroxy-5β-androsterone

40 U/ml 3α-hydroroxysteroid dehydrogenase

Sample

5, 10, 25, 50, 100 and 250 pg/ml horseradish peroxidase (finalconcentration: 2.5, 5, 12.5, 25, 50 and 125 pg/ml)

Method for Assay

To a flat-bottomed microplate well, 50 μl of a phosphate buffer solution(pH 7.0) containing the standard substance (horseradish peroxidase)(referred to as HRP below.) in a range of 0 to 250 pg/ml was added, andsubsequently, to the well, 50 μl of the reaction test solution wasadded, respectively, and the absorbance was measured for 120 minutesusing a 405 nm filter with a microplate reader (MTP-500 manufactured byCORONA CORPORATION) while warming the microplate to 37° C. The amount ofthe absorbance change for 120 minutes was plotted with respect to theconcentrations of the standard substance. The obtained straight linewell-dependent on the concentration is shown in FIG. 22.

Example 15 Assay of Pumilio by One Step Method Using3α-tert-butyl-peroxy-5β-androsterone

Reaction Test Solution

0.1M phosphate buffer solution (pH 7.0)

10 mM MgCl₂

6 mM thio-NAD

1 mM NADH

0.1 mM 3α-tert-butyl-peroxy-5β-androsterone

40 U/ml 3α-hydroxysteroid dehydrogenase

Sample

0.05, 0.1, 0.25, 0.5, 1 and 2.5 ng/ml Pumilio

Method for Assay

To a microplate immobilized with the anti-Pumilio guinea pig polyclonalantibody prepared with the method of Reference Example 5, 50 μl of TBS(pH 7.5) containing 0.1% BSA solution containing purified Pumilio (thestandard substance) in a range of 0 to 2.5 ng/ml was added, and themicroplate was stirred at room temperature for 2 hours. Then, thesolution in the well was removed with suction, and then the well waswashed with TBS (pH 7.5) containing 0.5% Triton X-100. 50 μl of TBS (pH7.5) containing 0.1% BSA solution containing the HRP-labeledanti-Pumilio mouse monoclonal antibody in a concentration ofapproximately 0.1 μg/ml was added, and the microplate was stirred atroom temperature for 1 hour. The solution in the well was removed withsuction, and then the well was washed with TBS (pH 7.5) containing 0.5%Triton X-100. Then, to each well, 50 μl of the reaction test solutionwas added, respectively, and the absorbance was measured for 60 minutesusing a 405 nm filter with a microplate reader (MTP-500 manufactured byCORONA CORPORATION) while warming the microplate to 37° C. The amount ofthe absorbance change for 60 minutes was plotted with respect to theconcentrations of the standard substance. The obtained straight linewell-dependent on the concentration is shown in FIG. 23.

Reference Example 9 Synthesis of 3α-benzoyl-peroxy-5β-androsterone and3α-acetyl-peroxy-5β-androsterone

The titled compound was obtained similarly to the synthesis of3α-tert-butyl-peroxy-5β-androsterone described in Example 14 except thattert-butyl-hydroperoxide was changed to benzoyl-hydroperoxide oracetyl-hydroperoxide.

INDUSTRIAL APPLICABILITY

The present invention can be suitably used in the field of clinicalexamination or the field of food examination that demands highsensitivity and simple measurement.

The invention claimed is:
 1. A method of assaying an enzyme activitycomprising the steps of: (a) providing an antibody-enzyme complex,wherein the enzyme is alkaline phosphatase, glucosidase, galactosidase,fructosidase, mannosidase or peroxidase (b) providing a substrate if theenzyme, wherein the substrate is an androsterone derivative representedby the following formula (1) that is a3α-hydroxy-17β-methoxy-5β-androstane derivative,

wherein: (i) in the 3α-hydroxy-17β-methoxy-5β-androstane derivative, Xrepresents a phosphate group, Y¹ represents hydrogen, and Y² represent amethoxy group when the 3α-hydroxy-17β-methoxy-5β-androstane derivativerepresented by the formula (1) is used as a substrate of alkalinephosphatase, (ii) in the 3α-hydroxy-17β-methoxy-5β-androstanederivative, X represents a sugar moiety, the sugar moiety represents oneselected from the group consisting of glucose, galactose, fructose andmannose, and Y¹ represents hydrogen, and Y² represents a methoxy group,when the 3α-hydroxy-17β-methoxy-5β-androstane derivative represented bythe formula (1) is used as a substrate of glucosidase, galactosidase,fructosidase or mannosidase, or (iii) in the3α-hydroxy-17β-methoxy-5β-androstane derivative, X represents —O—CO—R(provided that R represents a C₁₋₆ alkyl group or a phenyl group), Y¹represents hydrogen, and Y² represents a methoxy group; when the3α-hydroxy-17β-methoxy-5β-androstane derivative represented by theformula (1) is used as a substrate of peroxidase and (c) reacting theenzyme of the antibody-enzyme complex with the androsterone derivativeof formula (1) to produce a product; and (d) quantifying the product ofthe enzyme reaction (c) by producing thio-NADH and/or thio-NADPH byenzyme cycling reaction using NADH and/or NADPH, thio-NAD and/orthio-NADP, and hydroxysteroid dehydrogenase (HSD), wherein a substrateof the HSD is the product of the enzyme reaction (c), and assaying theamount of the produced thio-NADH and/or thio-NADPH, or measuring thechange of the color by the produced thio-NADH and/or thio-NADPH in orderto assay the enzyme activity.
 2. The method according to claim 1,wherein the enzyme is peroxidase, and X represents —O—CO—R (providedthat R represents a C₁₋₆ alkyl group or a phenyl group), and Y¹represents hydrogen, and Y² represents a methoxy group in the3α-hydroxy-17β-methoxy-5β-androstane derivative represented by theformula (1).
 3. A method of assaying a nucleic acid probe comprising thestep of (a) providing an enzyme-labeled nucleic acid probe, wherein theenzyme is alkaline phosphatase, glucosidase, galactosidase,fructosidase, mannosidase or peroxidase, (b) providing the substrate ofthe enzyme, wherein the substrate is an androsterone derivativerepresented by the following formula (1) that is a3α-hydroxy-17β-methoxy-5β-androstane derivative is a substrate of theenzyme,

wherein (i) in the 3α-hydroxy-17β-methoxy-5β-androstane derivative, Xrepresents a phosphate group, Y¹ represent hydrogen, and Y² represents amethoxy group when the 3α-hydroxy-17β-methoxy-5β-androstane derivativerepresented by the formula (1) is used as a substrate of alkalinephosphatase, (ii) X represents a sugar moiety, the sugar moietyrepresents one selected from the group consisting of glucose, galactose,fructose and mannose, and Y¹ represents hydrogen, and Y² represents a C₁methoxy group when the 3α-hydroxy-17β-methoxy-5β-androstane derivativerepresented by the formula (1) is used as a substrate of glucosidase,galactosidase, fructosidase or mannodidase, or (iii) X represents—O—CO—R (provided that R represents a C₁₋₆ alkyl group or a phenylgroup), Y¹ represents hydrogen, and Y² represents a methoxy group whenthe 3α-hydroxy-17β-methoxy-5β-androstane derivative represented by theformula (1) is used as a substrate of peroxidase, and (c) reacting theenzyme of the enzyme-labeled nucleic acid probe with the androsteronederivative of formula (1) to produce a product; (d) quantifying theproduct of the enzyme reaction (c) by producing thio-NADH and/orthio-NADPH by enzyme cycling reaction using NADH and/or NADPH, thio-NADand/or thio-NADP, and hydroxysteroid dehydrogenase (HSD), wherein asubstrate of the HSD is the reaction product of the enzyme of theenzyme-labeled nucleic acid probe and assaying the amount of theproduced thio-NADH and/or thio-NADPH, or measuring the change of thecolor by the produced thio-NADH and/or thio-NADPH.
 4. The methodaccording to claim 1, wherein the enzyme is alkaline phosphatase, and Xrepresents a phosphate group, Y¹ represents hydrogen, and Y² representsa methoxy group, in the 3α-hydroxy-17β-methoxy-5β-androstane derivativerepresented by the formula (1).
 5. The method according to claim 1,wherein the enzyme is glucosidase, galactosidase, fructosidase ormannosidase, and X represents a sugar moiety, the sugar moietyrepresents one selected from the group consisting of glucose, galactose,fructose and mannose, and Y¹ represents hydrogen, and Y² represents amethoxy group in the 3α-hydroxy-17β-methoxy-5β-androstane derivativerepresented by the formula (1).